Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays

ABSTRACT

This invention relates to detection of specific extracellular nucleic acid in human or animal blood plasma or serum associated with disease. Specifically, the invention relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA, and to methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated DNA found in blood plasma or serum. In particular, the invention relates to the detection, identification, or monitoring of the existence, progression or clinical status of neoplasia in humans or other animals that contain a mutation that is associated with the neoplasm through detection of the mutated nucleic acid of the neoplasm in plasma or serum fractions.

This application is a divisional of U.S. Ser. No. 10/298,816, filed Nov.18, 2002, now U.S. Pat. No. 6,939,675, issued on Jul. 31, 2003, which isa continuation of U.S. Ser. No. 09/642,952, filed Aug. 21, 2000, nowU.S. Pat. No. 6,521,409, which is a continuation of U.S. Ser. No.08/818,058, filed Mar. 14, 1997, now U.S. Pat. No. 6,156,504, which is acontinuation-in-part of U.S. Provisional Application, Ser. No.60/028,180, filed Oct. 15, 1996, and U.S. Provisional Application, Ser.No. 60/026,252, filed Sep. 17, 1996, and U.S. Provisional Application,Ser. No. 60/013,497, filed Mar. 15, 1996, each of which provisionalapplications is now abandoned, the entire disclosure of each of which ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for detecting specific extracellularnucleic acid in plasma or serum fractions of human or animal bloodassociated with neoplastic, pre-malignant or proliferative disease. Theinvention permits the detection of extracellular, tumor-associatednucleic acid in the serum or plasma of humans or other animalsrecognized as having a neoplastic, pre-malignant or proliferativedisease or in individuals without any prior history or diagnosis ofneoplastic, pre-malignant or proliferative disease. Specifically, theinvention relates to detection of nucleic acid derived from mutantoncogenes or other tumor-associated DNA, and to methods of detecting andmonitoring extracellular mutant oncogenes or tumor-associated DNA foundin the plasma or serum fraction of blood using DNA enrichment methods,wherein enrichment-based extraction methods are used prior toamplification and/or detection, or wherein enrichment for the nucleicacid of interest occurs during amplification, in particular through useof a restriction endonuclease. In particular, the invention relates tothe detection, identification, or interference of the existence ofpremalignant neoplasms or tissue in humans or other animals withoutcancer, wherein the neoplasm contains a mutation that is associated withthe neoplasm, through detection of the mutated nucleic acid associatedwith the neoplasm in plasma or serum fractions. The invention providesthe ability to detect extracellular nucleic acid derived from geneticsequences known to be associated with neoplasia, such as oncogenes, aswell as genetic sequences previously unrecognized as being associatedwith neoplastic, pre-malignant or proliferative disease. The inventionthereby provides methods for early identification of colorectal,pancreatic, lung, breast, bladder, ovarian, lymphoma and othermalignancies carrying tumor-related mutations of DNA and methods formonitoring cancer and other neoplastic disorders in humans and otheranimals.

2. Description of the Related Art

Neoplastic disease, including most particularly that collection ofdiseases known as cancer, is a significant part of morbidity andmortality in adults in the developed world, being surpassed only bycardiovascular disease as the primary cause of adult death. Althoughimprovements in cancer treatment have increased survival times fromdiagnosis to death, success rates of cancer treatment are more closelyrelated to early detection of neoplastic disease that enable aggressivetreatment regimes to be instituted before either primary tumor expansionor metastatic growth can ensue. A particularly favorable prognosis isachieved if premalignant tissue can be eradicated prior to progressionto cancer.

Oncogenes are normal components of every human and animal cell,responsible for the production of a great number and variety of proteinsthat control cell proliferation, growth regulation, and cell death.Although well over one hundred oncogenes have been described to datewith nearly all identified at the deoxyribonucleic acid (DNA) sequencelevel. It is likely that a large number of oncogenes remain to bediscovered.

Mutations in DNA occur as the result of inborn (inherited) geneticerrors or acquired (somatic) mutations, often as a result ofenvironmental insults, and have long been recognized as playing acausative role in the development of neoplastic disease. Within the lasttwenty years the sites of such mutations have been recognized to bewithin oncogenes, including tumor suppressor genes, and similartumor-related gene regions including those with DNA microsatellitealterations and hypermethylated genes. Mutation of these genes have beenfound to be an intrinsic and crucial component of premalignant andmalignant growth in both animals and humans. Many malignant tumors orcell lines derived from them have been shown to contain one or moremutated oncogenes, and it is possible that every tumor contains at leastone mutant oncogene.

Mutated oncogenes are therefore markers of malignant or premalignantconditions. It is also known that other, non-oncogenic portions of thegenome may be altered in the neoplastic state. Nucleic acid based assayscan detect both oncogenic and non-oncogenic DNA, whether mutated ornon-mutated.

In particular, nucleic acid amplification methods (for example, thepolymerase chain reaction) allow the detection of small numbers ofmutant molecules among a background of normal ones. While alternatemeans of detecting small numbers of tumor cells (such as flow cytometry)have generally been limited to hematological malignancies (Dressler andBartow, 1989, Semin. Diag. Pathol. 6: 55-82), nucleic acid amplificationassays have proven both sensitive and specific in identifying malignantcells and for predicting prognosis following chemotherapy (Fey et al.,1991, Eur. J. Cancer 27: 89-94).

Various nucleic acid amplification strategies for detecting smallnumbers of mutant molecules in solid tumor tissue have been developed,particularly for the ras oncogene (Chen and Viola, 1991, Anal. Biochem.195: 51-56; Kahn et al., 1991, Oncogene 6: 1079-1083; Pellegata et al.,1992, Anticancer Res. 12: 1731-1736; Stork et al., 1991, Oncogene 6:857-862). For example, one sensitive and specific method identifiesmutant ras oncogene DNA on the basis of failure to cleave a restrictionsite at the crucial 12th codon (Kahn et al., 1991, ibid.). Similarprotocols can be applied to detect any mutated region of DNA in aneoplasm, allowing detection of other oncogene DNA or tumor-associatedDNA. Since mutated DNA can be detected not only in the primary cancerbut in both precursor lesions and metastatic sites (Dix et al., 1995,Diagn. Molec. Pathol. 4: 261-265; Oudejans et al., 1991, Int. J. Cancer49: 875-879), nucleic acid amplification assays provide a means ofdetecting and monitoring cancer both early and late in the course ofdisease.

While direct analysis of neoplastic tissue is frequently difficult orimpossible (such as in instances of occult, unrecognized disease),peripheral blood is easily accessible and amenable to nucleic acid-basedassays such as those mentioned above. Many studies have used nucleicacid amplification assays to analyze the peripheral blood of patientswith cancer in order to detect intracellular DNA extracted fromcirculating cancer cells in patients, including one study which detectedthe intracellular ras oncogene from circulating pancreatic cancer cells(Tada et al., 1993, Cancer Res. 53: 2472-4). However, it must beemphasized that these studies attempt to use nucleic acid-basedamplification assays to detect extracted intracellular DNA withincirculating cancer cells. The assay is performed on the cellularfraction of the blood from patients having cancer using the cell pelletor cells within whole blood, and the serum or plasma fraction isconventionally ignored or discarded prior to analysis. Since such anapproach requires the presence of metastatic circulating cancer cells(for non-hematologic tumors), it is of limited clinical use in patientswith early cancers, and it is not useful in the detection ofnon-hematologic non-invasive neoplasms or pre-malignant states.

It was known in the prior art that small but significant amounts ofnormal DNA circulate in the blood of healthy people (Fedorov et al.,1986, Bull. Exp. Biol. Med. 102: 1190-2; Leon et al., 1977, Cancer Res.37: 646-50), and this amount has been found to increase in cancer states(Shapiro et al., 1983, Cancer 51: 2116-20; Stroun et al., 1989, Oncology46: 318-322). The prior art contains disclosure that mutant oncogene DNAcould be detected in peripheral blood plasma or serum of cancer patients(see, for example, Sorenson et al., 1994, Cancer Epidemiology,Biomarkers & Prevention 3: 67-71; Vasioukhin et al., 1994, Br. J.Haematol. 86: 774-9; Vasyukhin et al., in Verna & Shamoo (eds),Biotechnology Today, Ares-Serono Symposia Publications, pp. 141-150).Mutant ras oncogenes have been demonstrated in plasma or serum usingpolymerase chain reaction. However, these reports have also beengenerally limited to patients with advanced cancer or known neoplasticor proliferative disease.

We have recognized that nucleic acid amplification assays can detecttumor-associated extracellular mutated DNA, including oncogene DNA, inthe plasma or serum fraction of blood of humans withoutclinically-diagnosed cancer or known disease (see U.S. Ser. No.08/818,058, incorporated by reference), and that this can beaccomplished in a clinically useful manner.

SUMMARY OF THE INVENTION

The present invention relates to detection of specific extracellularnucleic acid in plasma or serum in human or animals without cancer whichare associated with neoplastic, preneoplastic or proliferative diseaseor conditions. Specifically, the invention relates to the detection ofpremalignant disease by the direct enrichment of mutated nucleic acid ortumor-associated nucleic acid found in the plasma or serum fraction ofblood with respect to wild-type or non-mutated nucleic acid either priorto or during amplification and detection of the nucleic acid, wherebythe concentration of the mutant nucleic acid is increased or the mutantnucleic acid is isolated from the remaining non-mutated nucleic acid.The invention thus provides methods for detecting predictive riskmarkers for a variety of cancers, including colorectal, pancreatic,lung, prostate, esophageal, gastric, breast, bladder, ovarian, cervical,liver, and kidney cancer, and other malignancies and premalignantconditions carrying tumor-associated mutations in DNA, as well asmethods for monitoring neoplastic disorders in humans and animals. Theinvention provides methods for detecting mutant oncogenes, including butnot limited to mutated K-ras, APC, and P53 DNA in plasma or serum.Premalignant diseases or conditions include but are not limited tocolorectal adenoma, cervical dysplasia, atypical squamous metaplasia ofthe lung, bronchial dysplasia, atypical hyperplasia of the breast,prostatic intraepithelial neoplasia, atypical endometrial hyperplasia,dysplastic nevi of the skin, and Barrett's esophagus.

The prior art provides instruction in identifying tumor-associated DNAin the plasma or serum fraction of blood of humans with known malignancyusing DNA extraction methods, followed by amplification of the targetDNA, followed by detection of the amplified target nucleic acid (see,for example, Sorenson et al., 1993, American Association for CancerResearch Abstract #174; Vasyukhin et al., 1994, Biotechnology Today(Verna & Shamoo, eds.), Ares-Serono Symposia Publications, pp. 141-150;Vasioukhin et al., 1994, Br. J. Haematol. 86: 774-9; Lefort et al.,1995, American Association for Cancer Research Abstract #557; Nawroz etal., 1996, Nature Med. 2:1035-7; Chen et al., 1996, Nature Med. 2:1033-5). However, the prior art does not apply these methods tonon-hematologic premalignancy, or to patients without known disease. Themethods disclosed herein, in contrast, allow detection of mutant DNA ortumor-associated DNA from the blood of humans without cancer or knowndisease by providing for the enrichment of mutated nucleic acid, whereinthe mutated nucleic acid is concentrated and/or isolated from theremaining extracted nucleic acid prior to or independent ofamplification of the target nucleic acid, and thereby provides methodswhich enable enhanced detection of the target nucleic acid or a fragmentthereofs. The methods disclosed herein further provide for theenrichment of mutated nucleic acid with respect to wild-type nucleicacid during amplification of the target nucleic acid, and therebyprovide for methods which enable the enhanced detection of the targetnucleic acid or a fragment thereofs.

Extracellular DNA is known to circulate in the serum or plasma fractionof blood (Stroun et al., 1987, Eur. J. Cancer Clin. Oncol. 23: 707-12;Stroun et al., 1989, Oncology 46: 318-322). The invention disclosed inco-owned and co-pending U.S. Ser. No. 08/818,058 (incorporated byreference) taught methods for identifying mutated extracellular DNA foridentification of premalignant lesions. These methods are useful fordiagnosis and treatment of people who are at risk to develop malignancyor premalignancy. Appropriate therapy, including but not limited to,increased surveillance, surgical excision, chemotherapy andimmunotherapy or chemoprevention therapies, as well as more innovativetherapies (such as antisense oligonucleotide therapy directed at amutated KRAS oncogene or vaccine therapy, for example), may beinstituted based on detection of mutant or non-mutated tumor-associatednucleic acid in such patients.

Prior to the instant invention and co-owned and co-pending U.S. Ser. No.08/818,058 it was not known that extracellular mutant nucleic acid couldbe detected in the blood of humans without cancer. The present inventionand co-owned and co-pending U.S. Ser. No. 08/818,058 teach thatdetection of extracellular mutant nucleic acid in the blood of humanswithout cancer can be enhanced by enrichment of the mutated nucleic acidrelative to wild-type nucleic acid wherein the mutated nucleic acid isconcentrated and/or isolated from the remaining extracted nucleic acidin a manner performed either concurrently or sequentially with theextraction and prior to amplification and/or detection of the targetnucleic acid or a fragment thereof. Although extracellular mutanttumor-associated DNA has been detected in patients with advancedmalignancies (Sorenson et al., 1993, Id.; Vasioukhin et al., 1994, Id.;Nawroz et al., 1996, Id.), it had been presumed in the prior art thatonly malignancies, and in particular large malignancies, produced enoughextracellular DNA to be identified even with prior amplification of theDNA. Co-owned and co-pending U.S. Ser. No. 08/818,058 (incorporated byreference) taught that mutated oncogenes and other tumor-associatednucleic acid was also detectable in blood serum or plasma of individualswith pre-malignant lesions, diseases, or conditions followingamplification of nucleic acid sequences found in blood plasma or serum.In the present invention additional methods are taught which enableenrichment of mutated nucleic acid thereby providing enhanced detectionof nucleic acid or a fragment thereof whereby detection is indicative orassociated with the presence of premalignant tissue in the human.

The present invention provides methods for detecting the presence ofextracellular nucleic acid in blood plasma or serum fractions, saidnucleic acid being associated with a neoplastic, pre-malignant orproliferative disease state in an animal or a human without cancer. Theinvention provides methods for extracting and enriching extracellularnucleic acid associated with a neoplastic, pre-malignant orproliferative disease state in an animal or a human prior to nucleicacid amplification or signal detection. The invention further providesfor methods whereby mutated nucleic acid is enriched with respect towild-type nucleic acid during or prior to an amplification step whereina restriction endonuclease is used during or prior to the amplificationstep. These methods of the invention are used for the detecting,monitoring, evaluating, or risk assessment of premalignant conditions,and in particular those conditions including but not limited tocolorectal adenoma, cervical dysplasia, atypical squamous metaplasia ofthe lung, bronchial dysplasia, atypical hyperplasia of the breast,prostatic intraepithelial neoplasia, atypical endometrial hyperplasia,dysplastic nevi of the skin, and Barrett's esophagus.

In a first aspect, the invention provides a method for detectingextracellular tumor-derived or tumor-associated mutated nucleic acid ina plasma or serum fraction of a blood sample from a human or animalwithout cancer, thereby providing a method for diagnosis, detection,monitoring, evaluation or treatment of a neoplastic or proliferativedisease in an animal or a human. The method provided by the inventioncomprises the steps of: first, purifying extracellular nucleic acid fromblood plasma or serum to prepare a preparation of extracted nucleic acidcontaining a tumor-associated mutated nucleic acid or a fragmentthereof; second, enriching for the mutated nucleic acid, eitherconcurrent with or sequentially following the initial extraction step,wherein the mutated nucleic acid or a fragment thereof is concentratedand/or isolated from the remaining extracted nucleic acid; third,amplifying the enriched mutated nucleic acid or a fragment thereof, oramplifying a signal corresponding to the enriched mutated nucleic acidor a fragment thereof; and fourth, detecting the product of theamplified mutated nucleic acid or a fragment thereof, or the amplifiedsignal corresponding to the extracted mutated nucleic acid or a fragmentthereof, wherein the mutated nucleic acid or a fragment thereof isassociated with neoplastic, pre-malignant or proliferative disease. Inpreferred embodiments of this aspect of the invention, the mutatednucleic acid is derived from nucleic acid encoding a mutated oncogene orother tumor-associated DNA, such as a DNA microsatellite alteration.

In another aspect, the invention provides a method for detectingextracellular tumor-derived or tumor-associated nucleic acid in a plasmaor serum fraction of a blood sample from a human or animal withoutclinically-diagnosed cancer, thereby providing a method for detection,diagnosis, monitoring, evaluation, or treatment of a neoplastic orproliferative disease or premalignant conditions in an animal or ahuman. The method provided by the invention comprises the steps of:first, purifying extracellular nucleic acid from plasma or serum toprepare a preparation of extracted nucleic acid containing atumor-associated mutated nucleic acid or a fragment thereof; second,enriching the mutated nucleic acid relative to wild-type nucleic acidusing an endonuclease prior to or during amplification; third,amplifying the enriched mutated acid or a fragment thereof; and fourth,detecting the amplified fragment of the mutated nucleic acid or a signalcorresponding to the amplified fragment of the mutated nucleic acid. Inpreferred embodiments of this aspect of the invention, the nucleic acidis derived from a nucleic acid encoding an oncogene or othertumor-associated DNA.

Particularly preferred embodiments of the invention comprise detectionof nucleic acid sequences derived from or related to mutated p53, K-ras,and APC alleles.

In preferred embodiments of the inventive methods, extracellular nucleicacid is extracted from blood plasma or serum using an extraction methodincluding gelatin extraction; silica, glass bead, or diatom extraction;guanidine- or guanidinium-based extraction; chemical extraction methods;and size-exclusion and anion-exchange chromatographic methods. Inparticularly preferred embodiments, the target DNA is extracted in anenriching manner, or extracted DNA is further enriched, usingprobe-specific hybridization wherein said hybridizing probes areimmobilized to a substrate, wherein such substrate includes but is notlimited to nylon and magnetic beads, from which contaminating species(nucleic acid and otherwise) can be removed using methods (such aswashing at defined stringencies of salt concentration and temperature)known in the prior art, or wherein alternatively the target DNA ofinterest may be otherwise isolated from contaminating species includingwild-type DNA, for example by application of a magnetic field or anelectric field. In preferred embodiments, the extracted and enrichednucleic acid is amplified or signal amplified, wherein the amplificationmethod may include but is not limited to polymerase chain reaction,ligase chain reaction, boomerang DNA amplification, strand displacementamplification, strand displacement activation, cycling probeamplification, and branched DNA signal amplification. In preferredembodiments, DNA detection is performed using a detection methodincluding gel electrophoresis; immunological detection methods;hybridization using a specific, fluorescent-, radioisotope-, antigenic-or chromogenically-labeled probe; Southern blot analysis;electrochemiluminescence; reverse dot blot detection; andhigh-performance liquid chromatography.

In a preferred embodiment, the nucleic acid is extracted from bloodserum or plasma by heating the serum or plasma, at a temperature fromabout 90° C. to about 100° C., more preferably from about 95° C. toabout 100° C., for a time from about 1 minute to about 20 minutes, morepreferably from about 5 minutes to about 15 minutes, and most preferablyfrom about 5 minutes to about 10 minutes. Optionally, the blood plasmaor serum can be frozen after boiling to a temperature of from about −20°C. to about 0° C. for at least about 5 minutes, more preferably 15minutes and most preferably for at least about 30 minutes. The boilingblood plasma or serum is used after cooling or, if frozen, after beingthawed to a liquid.

The methods of the invention are provided for detecting tumor-associatedextracellular nucleic acid, for example a mutated oncogene, in a humanwithout clinically-diagnosed cancer, whereby detection is indicative ofthe presence of non-hemopoietic cells or tissue containingtumor-associated nucleic acid within the human, the method comprisingthe steps of purifying extracellular nucleic acid from a plasma or serumfraction of a blood sample from the human to prepare extracted nucleicacid containing a tumor-associated nucleic acid, for example a mutatedoncogene DNA, or a fragment thereof, and concurrently or sequentially;enriching for the tumor-associated nucleic acid or a fragment thereof,wherein the tumor-associated nucleic acid or a fragment thereof isconcentrated and/or isolated from the remaining extracted nucleic acid;and amplifying or signal amplifying the tumor-associated nucleic acid ora fragment thereof; and detecting the product of the amplifiedtumor-associated nucleic acid or a fragment thereof or the amplifiedsignal of the tumor-associated nucleic acid or a fragment thereof. Thedetected nucleic acid or a fragment thereof is then identified, e.g., ascomprising the mutated form of an oncogene associated with a neoplastic,pre-malignant or proliferative disease, wherein detection of theamplified product or the amplified signal or the tumor-associatednucleic acid or a fragment thereof is indicative of the presence ofnon-hemopoietic cells or tissue having a mutated oncogene or othertumor-associated nucleic acid in the human. In a preferred embodiment,the methods of the invention are used as an aid in the diagnosis in ahuman of a neoplastic, pre-malignant or proliferative disease. Inanother preferred embodiment, the method is used to determine apredictive risk factor for a neoplastic disease or disease progressionin a human. Additionally, the methods of the invention are preferablyused to determine disease prognosis in a human. In other preferredembodiments, the methods of the invention are used to determine the needfor additional diagnostic tests, or for treatment.

Particularly preferred methods of the invention enable detection inblood of mutated nucleic acid, wherein the mutation is an acquired(somatic) mutation, as may be optionally shown by demonstrating theabsence of the mutation in normal cells from the human having theacquired mutation, such as in normal leukocytes or other normal tissue.The invention thus provides a method for determining an acquiredpredictive risk factor for a non-hematologic disease in a human withoutclinically-diagnosed cancer, the method comprising the steps ofpurifying extracellular nucleic acid from blood or blood plasma or serumfrom a human without clinically-diagnosed cancer to prepare extractednucleic acid containing a mutated DNA or a mutated DNA fragment, andconcurrently or sequentially enriching for the mutated DNA or a fragmentthereof, wherein the mutated DNA or a fragment thereof is concentratedand/or isolated from the remaining extracted nucleic acid, andthereafter amplifying the enriched mutated DNA or a fragment thereof, oralternatively amplifying a signal from the enriched mutated DNA or afragment thereof, and then detecting the product of the amplifiedmutated DNA or the product of its amplified fragment, or the amplifiedsignal of the mutated DNA or the amplified signal of a fragment thereof,whereby said detection determines a predictive risk factor for anon-hematologic disease. Further, and optionally, by demonstrating theabsence of the mutated DNA in normal cells of the human, such as normalleukocytes or other normal tissue, in which it is therefore demonstratedthat the mutation is not an inherited or inborn mutation, the predictiverisk factor is shown to be an acquired predictive risk factor. In apreferred embodiment the mutated DNA includes but is not limited toeither a mutated oncogene DNA, for example mutated K-ras, P53, or APC,or a DNA microsatellite alteration. In preferred embodiments thenon-hematologic disease is either colorectal adenoma, cervicaldysplasia, atypical squamous metaplasia of the lung, bronchialdysplasia, atypical hyperplasia of the breast, prostatic intraepithelialneoplasia, atypical endometrial hyperplasia, dysplastic nevi of theskin, or Barrett's esophagus.

Also provided as embodiments of the methods of the invention are methodsadditionally comprising the steps of determining the nucleic acidsequence of the nucleic acid fragment of extracellular nucleic acid inthe extracted nucleic acid fraction that is associated with neoplasticor proliferative disease, wherein the nucleic acid sequence of thenucleic acid fragment comprises a mutated or variant allele of a nucleicacid associated with a neoplastic or proliferative disease.

In addition to the methods noted above, the invention provides methodsfor isolating extracellular tumor-derived or tumor-associated nucleicacid from a fraction of a blood sample comprising the plasma fraction orthe serum fraction of the blood sample. In these embodiments the methodcomprises the steps of purifying extracellular nucleic acid from plasmaor serum to prepare a preparation of extracted nucleic acid using anextraction method; enriching the extracted nucleic acid fraction for theportion of the fraction that is associated with neoplastic,pre-malignant or proliferative disease; and cloning the DNA fragmentscomprising the enriched nucleic acid fraction that is associated withneoplastic, pre-malignant or proliferative disease. Also provided inthis aspect of the invention are recombinant genetic constructscomprising a nucleic acid fragment that is associated with a neoplastic,pre-malignant or proliferative disease or condition that is preparedusing the methods of the invention. Ribonucleic acid transcribed fromthe recombinant genetic constructs of the invention are also provided,as well as protein produced from translation of said RNA, and methodsfor using the translated proteins and peptides of the invention asepitopes for the production of antibodies and vaccines.

The invention also provides a method for concentrating and/or isolatingany extracellular mutated DNA or a fragment thereof present in plasma orserum from a human without clinically-diagnosed cancer for whichspecific oligonucleotide hybridization primers are available, the methodcomprising the steps of purifying extracellular nucleic acid from bloodof a human without clinically-diagnosed cancer to prepare nucleic acidcontaining a mutated DNA or a mutated DNA fragment, and concurrently orsequentially hybridizing an oligonucleotide to the mutated DNA or amutated DNA fragment to produce a hybridized product of mutated DNA or afragment thereof, and separating the hybridized product of mutated DNAor a fragment thereof from the remaining non-hybridized nucleic acid,thereby concentrating and/or isolating the mutated DNA or a fragmentthereof. The invention thus provides a method for enriching for amutated oncogene allele in plasma or serum, including but not limited tomutated K-ras, P53, or APC, and enriching for a DNA having amicrosatellite alteration. In a preferred embodiment, the signal fromthe specific nucleic acid fragments comprising the enriched nucleic acidfraction is amplified using methods known to those with skill in theart, for example, branched DNA signal amplification, and combinations orvariations thereof. In a preferred embodiment, detection of specific DNAfragments is performed using a detection method such as gelelectrophoresis, immunological detection methods, nucleic acidhybridization using specific, fluorescent- or chromogenically-labeledprobe, Southern blot analysis, electrochemiluminescence, reverse dotblot detection, or high-performance liquid chromatography.

The invention further provides a method for detecting a mutation in theDNA present in blood or a blood fraction, i.e., plasma or serum, of ahuman having a disease or premalignant condition in whom the presence ofthe disease or premalignant condition is unrecognized, wherein themutation is an acquired mutation of known association with the diseaseor premalignant condition, and wherein the disease or premalignantcondition includes but is not limited to colorectal adenoma, cervicaldysplasia, atypical squamous metaplasia of the lung, bronchialdysplasia, atypical hyperplasia of the breast, prostatic intraepithelialneoplasia, atypical endometrial hyperplasia, dysplastic nevi of theskin, or Barrett's esophagus. The method comprises the steps ofpurifying extracellular nucleic acid from blood or a blood fraction froma human in whom the presence of a disease or premalignant condition isunrecognized to prepare extracted nucleic acid containing a mutated DNAor a fragment thereof wherein the mutation is of known association withthe disease or premalignant condition, and amplifying the mutated DNA ora fragment thereof, or alternatively amplifying a signal from themutated DNA or a fragment thereof, and detecting the product of theamplified mutated DNA or a fragment thereof, or the amplified signal ofthe mutated DNA or a fragment thereof. In a particularly preferredembodiment, the method enables the evaluation of blood or a bloodfraction from a human for the mutated oncogene to assist in theidentification of the premalignant disease or premalignant condition.

It is therefore the object of this invention to detect or infer thepresence of precancerous cells from non-hematologic premalignancies,within a human or animal body having a recognized neoplastic orproliferative disease or in those not previously diagnosed, by examiningthe plasma or serum fraction of blood for extracellular mutated oncogeneDNA or tumor-derived or associated extracellular DNA, using a nucleicacid detection assay.

Another object of this invention is to enable extraction ofextracellular tumor-associated nucleic acid from plasma or serum in anenriching manner, wherein the tumor-associated nucleic acid isconcentrated and/or isolated from the remaining extracted nucleic acid.

An advantageous application of this invention is to identify, eitherquantitatively or qualitatively, acquired mutant oncogenes or acquiredtumor-associated DNA in the blood plasma or serum of humans or animalsas to classify such patients for their risk of neoplastic disease.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, mutant oncogenes ortumor-associated DNA in the blood plasma or serum of humans or animalswho are receiving therapies, including but not limited tochemopreventive therapy and polypectomy, as a guide to whether adequatetherapeutic effect has been achieved or whether additional or moreadvanced therapy is required, and to assess prognosis in these patients.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, mutant oncogenes ortumor-associated DNA in the blood plasma or serum of humans or animalsas to determine the need for additional diagnostic testing, whereinadditional diagnostic testing includes but is not limited to endoscopy,colonoscopy, sigmoidoscopy, bronchoscopy, radiologic imaging,radionucleotide scanning, ultrasonography, PET scanning, or furtherevaluation of organ or site specific bodily fluids or stool, whereinbodily fluid includes but is not limited to that collected by drainage,aspiration, direct sampling, and lavage.

Another advantageous application of this invention is to identify,either by detection or inference, the presence of premalignantneoplasms, conditions, or diseases, through detection of acquired mutantoncogenes or acquired tumor-associated DNA in the blood of humans oranimals in whom the presence of the neoplasm, condition, or disease isunrecognized, wherein the acquired mutant DNA derives from premalignantgrowths such as dysplasias or adenomas, or from other cells bearing anacquired mutated DNA of known association with a premalignant disease orcondition, wherein said diseases and conditions include but are notlimited to colorectal adenoma, cervical dysplasia, atypical squamousmetaplasia of the lung, bronchial dysplasia, atypical hyperplasia of thebreast, prostatic intraepithelial neoplasia, atypical endometrialhyperplasia, dysplastic nevi of the skin, and Barrett's esophagus. Inaddition, the invention advantageously provides a panel of severaloncogene assays that can distinguish malignant from premalignantconditions, or assist in medical monitoring to detect transformation ofthe growth to an outright malignancy, or to detect regression.Furthermore, the invention advantageously provides a means to definerisk of malignancy in a human wherein the risk was previouslyunrecognized. Thus, the invention provides methods for earlyidentification of neoplasms and premalignant conditions of the colon,rectum, pancreas, lung, breast, bladder, ovary, cervix, endometrium,liver, prostate, and stomach and of other neoplasms or premalignantconditions carrying tumor-related mutations of DNA, and methods formonitoring neoplastic disorders in humans and other animals.

Advantageous application of the invention is provided as example but notlimitation to include detection of mutated K-ras allele in blood from ahuman with adenoma; detection of mutated P53 allele in blood from ahuman with adenoma; detection of mutated APC allele in blood from ahuman with a colorectal cancer; detection of mutated K-ras allele inblood from a human with a proliferative disease; and detection ofmutated K-ras allele in blood from a human with an in-situ (non-invasiveor pre-invasive) carcinoma.

Thus, the invention provides a method of screening both healthyindividuals and individuals at risk for cancer and premalignantconditions.

Another advantageous application of this invention is to identify,either quantitatively or qualitatively, one or more of multiple mutantoncogenes or tumor-associated DNAs in the blood plasma or serum ofhumans or animals without clinically-diagnosed cancer or not known tohave cancer by use of a panel of oligonucleotide primers each specificfor one of multiple differing oncogene alleles detected by the panel,wherein a particularly preferred application employs a nucleic acidenrichment method, and wherein the oncogene alleles are detectedconcurrently or sequentially. Additionally, said panel enables theinference of the presence of specific tumor types based upon mutatedoncogenes or other tumor-associated mutated nucleic acid detected by thepanel. Additionally, said panel enhances identification of humans atrisk for neoplastic disease. Additionally, said panel enhancesidentification of humans having premalignant or malignant tissue. Aparticularly preferred advantageous application of this inventionidentifies one or more of either K-ras, P53, and/or APC mutated allelesin blood from a human without clinically-diagnosed cancer by use of apanel of primers specific for at least one or more of either K-ras, P53,or APC mutated alleles, whereby a human at risk for a gastrointestinalneoplastic disease is identified.

The invention thereby provides for methods of detecting an acquiredmutated APC allele or a fragment thereof in blood or a blood fraction.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods for detecting or inferring the presenceof precancerous cells in a human or animal. The method comprisespreparation of nucleic acid from plasma or serum wherein the extractionof nucleic acid may be performed with or without DNA precipitation orother separation of soluble nucleic acid from the plasma or serum, andfurther performed using a method of enrichment for mutated nucleic acid,wherein a mutated nucleic acid is concentrated and/or isolated, ineither case followed by detection of mutated nucleic acid preferentiallyfollowing amplification of the mutated nucleic acid or its signal.Enrichment of the mutated nucleic acid may occur prior to or duringamplification by methods provided, including methods using anendonuclease or by methods involving hybridization of the mutatednucleic acid. Embodiments of the invention which provide for enrichmentof mutated DNA from blood or blood fractions offer the beneficialadvantage of enhancing detection of the mutated DNA. Direct sequencingof mutant nucleic acid is further made possible so that a broad range ofmutated oncogenes including tumor suppressor genes, translocated genes,hypermethylated genes, microsatellite alterations in DNA, and non-codingDNA related to the development of malignancy may be identified.Embodiments of the invention provided that exhibit the absence of DNAprecipitation methods from the invention serves several beneficialfunctions. First, such methods reduce the manipulation required for eachsample, thus decreasing time and cost in preparation. Second, suchdecrease in manipulation reduces the risk of contamination of onespecimen with another or with exogenous DNA. Third, lack ofprecipitation steps decrease losses attendant with specimen handling andextraction, thus reducing the sample volume required and potentiallyincreasing the yield of DNA analyzed in each specimen. Fourth, suchmethods increase the ease with which the entire analytical process maybe automated, further reducing costs and turn-around time. Fifth, byreducing the variability in yield inherent in a precipitation process,such methods enable meaningful quantification of mutant nucleic acid,potentially providing prognostic information related to tumor burden.

The invention further provides a number of methods of identifyingmutations without prior knowledge of their location to be employed. Thissignificantly increases the utility of detecting mutated DNA in plasmaor serum, since genes such as APC and p53 exhibit point mutationsthroughout their coding regions (Hollstein et al., 1994, Nucleic AcidsRes. 22: 3551-5). This is a significant improvement over the prior art,which requires prior knowledge of the location of a mutation orrestriction of a search for mutants to the few most frequently mutatedloci (“hot spots”; see Sorenson et al., 1993, Id.; Vasioukhin et al.,1994, Id.; Nawroz et al., 1996, Id.; Sorenson et al., 1994, CancerEpidemiology, Biomarkers & Prevention 13: 67-71). Thus, a larger varietyof malignant and premalignant conditions may be assessed (sincedifferent tumor types seem to carry particular mutations not found inother tumor types) and they may be detected for more thoroughly. It isto be noted and understood that the methods of DNA preparation andenrichment described herein are furthermore applicable to the previouslydescribed aspects of the invention utilizing amplification as well asmethods of detection without amplification.

The invention provides methods for detection of an acquired (somatic)DNA mutation in the blood of a human without clinically-diagnosedcancer, wherein normal cells from the human lack the mutation; therebythe invention provides for a method of determining acquired predictiverisk factors for non-hematologic acquired diseases and conditions.

Moreover, the assays and methods of the invention can be performedqualitatively, whereby the amount of the nucleic acid product producedis at least sufficient for efficient detection of the product, orquantitatively, whereby the amount of the nucleic acid product producedis measured with reference to a standard useful in determining thesignificance of the amount of produced nucleic acid (for example,wherein the amount of nucleic acid product is related to a disease stateor risk of developing a disease state).

Specifically, the invention provides methods for detecting nucleic acidin plasma or serum of a human or animal without clinically-diagnosedcancer wherein the nucleic acid is associated with the existence ofpre-malignant cells or tissues in the human or animal, thereby providinga sensitive predictive risk factor for neoplastic disease andpremalignant conditions, wherein such diseases and conditions includebut are not limited to colorectal adenoma, cervical dysplasia, atypicalsquamous metaplasia of the lung, bronchial dysplasia, atypicalhyperplasia of the breast, prostatic intraepithelial neoplasia, atypicalendometrial hyperplasia, dysplastic nevi of the skin, and Barrett'sesophagus.

The invention further provides methods for identification of humanshaving unrecognized disease who might therefore benefit from additionaldiagnostic testing, including but not limited to colonoscopy,sigmoidoscopy, endoscopy, bronchoscopy, radiologic imaging including CTscans, spiral CT scans, MRI, contrast studies, and plain films,ultrasonography, radionucleotide imaging, PET scanning, and evaluationof organ specific bodily fluid, stool, or lavage fluid, wherein bodilyfluid includes but is not limited to that collected by drainage,aspiration, direct sampling, and lavage.

A General Overview of the Inventive Methods

In the practice of the invention blood is drawn by standard methods intoa collection tube, preferably comprising siliconized glass, eitherwithout anticoagulant for preparation of serum or with EDTA, heparin, orsimilar anticoagulants, most preferably EDTA, for preparation of plasma.Plasma may optionally be subsequently converted to serum by incubationof the anticoagulated plasma with an equal volume of 0.025 molar calciumchloride at 37° C. for a brief period, most preferably for 1-3 minutes,until clotting takes place. The clot may then be pelleted by a briefcentrifugation (1-10 seconds) at 1,000×g or greater, and thedeproteinized plasma removed to another tube. Preferably, the volume ofplasma used is 5 μL mixed with 5 μL of calcium chloride, then diluted to100 μL with water. Alternatively, the centrifugation may be omitted. Theserum or plasma may be utilized directly for identification of mutantDNA. In one preferred embodiment, 10 μL of serum or the prepared plasmais heated to a temperature greater than 90° C., most preferably greaterthan 94° C., for several minutes, most preferably 10. This heatedsubstrate may then be cooled to below room temperature for a period ofseveral minutes, or may be used directly in subsequent steps of theinvention. In either instance an optional step of brief centrifugationat 1,000×g or greater may be performed to pellet any precipitate.Alternatively, heating may take place by placing a volume of sample,preferably 5 μL, in a tube or microtiter well under mineral oil andheating in a microwave for greater than 3 minutes, more preferably 3-10minutes (as described in Sandford et al., 1997, Biotechniques 23:890-2). In most preferred embodiments, nucleic acid is extracted fromplasma or serum as an initial step of the invention. The extraction stepmay be performed either prior to concurrent with the enrichment step.

Gelatin Extraction Method

In a preferred embodiment, DNA is co-precipitated from plasma or serumwith gelatin by a method modified from that of Foumie et al. (1986,Anal. Biochem. 158: 250-256). A stock 5% (w/v) gelatin solution isprepared by mixing 1 gram gelatin (G8-500, Fisher, Pittsburgh, Pa.) with20 mL sterile, double-distilled water, autoclaving for 30 minutes, andfiltering through a 0.2 micron filter. The resultant solution issequentially frozen in a dry ice/ethanol bath and thawed at roomtemperature for a total of five cycles. A working 0.3% gelatin solutionis prepared by heating the stock solution to 60° C. and mixing 600 μL of5% gelatin with 25 μL of 1 M Tris-HCl (pH 8.0) and 9.4 mL of sterile,double-distilled water.

Plasma or serum (160 μL) is mixed with 12.8 μL of 0.5 M EDTA and 467 μLof sterile, double-distilled water, then emulsified for 3 minutes with320 μL of phenol or phenol:chloroform:isoamyl alcohol (25:24:1 ratio).The solution is centrifuged at 14,000×g for 10 minutes, and 570 μL ofthe aqueous layer is removed to a clean tube. DNA is precipitated byaddition of 142 μL of the 0.3% gelatin working solution and of 500 μL ofcold absolute ethanol, followed by incubation at −20° C. for 1-2 hours.Extracellular DNA may be precipitated within less than 1 h of incubationat −20° C., and a very short incubation may be preferable in somecircumstances. Alternatively, longer incubation at −20° C. for 1-2 hoursinsures the precipitation of most DNA. The sample is centrifuged at14,000×g at 4-6° C. for 15 minutes, washed once with cold 70% ethanol,and dried in a 60° C. heat block for 10 minutes. DNA is then recoveredby the addition of 35 to 70 μL of sterile, double-distilled waterpreheated to 60° C.

Glass Bead, Silica Particle, or Diatom Extraction Method

As an alternative rapid method of extracting extracellular DNA fromplasma or serum, glass beads, silica particles, or diatoms may be used,as in the method or adaptation of Boom et al. (Boom et al., 1991, J.Clin. Microbiol. 29: 1804-1811; Boom et al., 1989, J. Clin. Microbiol.28: 495-503). Size fractionated silica particles are prepared bysuspending 60 grams of silicon dioxide (SiO₂, Sigma Chemical Co., St.Louis, Mo.) in 500 mL of demineralized sterile double-distilled water.The suspension is then settled for 24 hours at room temperature.Four-hundred thirty (430) mL of supernatant is removed by suction andthe particles are resuspended in demineralized, sterile double-distilledwater added to a final volume of 500 mL. After an additional 5 hours ofsettlement, 440 mL of the supernatant is removed by suction, and 600 μLof HCl (32% wt/vol) is added to adjust the suspension to a pH 2. Thesuspension is aliquotted and stored in the dark.

Lysis buffer is prepared by dissolving 120 grams of guanidinethiocyanate (GuSCN, Fluka Chemical, Buchs, Switzerland) into 100 ML of0.1 M Tris hydrochloride (Tris-HCl) (pH 6.4), and 22 mL of 0.2 M EDTA,adjusted to pH 8.0 with NaOH, and 2.6 grams of Triton X-100 (PackardInstrument Co., Downers Grove, Ill.). The solution is then homogenized.

Washing buffer is prepared by dissolving 120 grams of guanidinethiocyanate (GuSCN) into 100 mL of 0.1 M Tris-HCl (pH 6.4).

50 μL of plasma or serum are mixed with 40 μL of silica suspensionprepared as above, and with 900 μL of lysis buffer, prepared as above,using an Eppendorf 5432 mixer over 10 minutes at room temperature. Themixture is then centrifuged at 12,000×g for 1 minute and the supernatantaspirated and discarded. The silica-DNA pellet is then washed twice with450 μL of washing buffer, prepared as above. The pellet is then washedtwice with 1 mL of 70% (vol/vol) ethanol. The pellet is then given afinal wash with 1 mL of acetone and dried on a heat block at 56° C. forten minutes. The sample is eluted for ten minutes at 56° C. with a TEbuffer consisting of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) with or withoutProteinas K (100 ng/ml) as described by Boom et al., ibid. Followingelution, the sample is then centrifuged at 12,000×g for three minutes,and the DNA-containing supernatant recovered (Boom et al., 1991, ibid.;Boom et al., 1989, ibid.; Cheung et al., 1994, J. Clin. Microbiol. 32:2593-2597).

Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction Method

As an alternative method, extracellular DNA may be extracted from plasmaor serum in one step using variations of the acid guanidiniumthiocyanate-phenol-chloroform extraction method. For example,extracellular DNA may be extracted from plasma or serum using TRIreagent, a monophase guanidine-thiocyanate-phenol solution, as describedby Chomczynski (1993, Biotechniques 15: 532-534). Plasma or serum (1 mL)is processed using 5-10 mL of TRI Reagent (commercially available as TRIReagent, Molecular Research Center, Cincinnati, Ohio, and from othersources) according to manufacturer's directions. DNA is precipitatedfrom the interphase with ethanol.

Other commercially available kits known to the art may be utilized toextract nucleic acid from plasma or serum providing purified DNA for usein the invention's first step, including Qiagen columns (QIAamp bloodkit, Qiagen, Basel, Switzerland), Boehringer Mannheim's High-Pure ViralNucleic Acid kit, and other commercial kits for extraction of nucleicacid from plasma or serum as described herein, which are provided asexample and not as limitation. Such kits may be used according tomanufacturer's directions.

Enrichment Methods

Extracellular nucleic acid can be enriched from plasma, serum or wholeblood using hybridization methods specific for particular species onextracellular nucleic acid. For example, nucleic acid derived frommutant K-ras oncogene DNA can be enriched from whole blood, serum orplasma using specific oligonucleotides for hybridization. Sucholigonucleotides, generally ranging in size from about 12 to about 15nucleotides or longer, are advantageously centered around the mutatednucleotide of interest to provide the greatest degree of discriminationbetween mutant and wildtype alleles. In particular, for the K-rasoncogene, the nucleotides of interest are the first and second positionsof codons 12, 13 and 61. For K-ras, codon 12, first position, exemplaryoligonucleotides have the sequence:GTTGGAGCTCGTGGCGTAG  (SEQ ID No.: 1)GTTGGAGCTTGTGGCGTAG  (SEQ ID No.: 2)andGTTGGAGCTAGTGGCGTAG,  (SEQ ID No.: 3)where the underlined nucleotide in each oligonucleotide is mutated fromthe wildtype.

In another example, many mutations associated with the development ofmalignancy have been identified in the tumor suppressor gene p53 from anumber of different tumors and tumor types. In colorectal carcinoma, forexample, the most common mutation involves codon 175. For this mutation,exemplary enrichment oligonucleotides include the following:CCATGAGCACTGCTCAG  (SEQ ID No.: 4)CCATGAGCTCTGCTCAG  (SEQ ID No.: 5)andCCATGAGCCCTGCTCAG,  (SEQ ID No.: 6)where the underlined nucleotide in each oligonucleotide is mutated fromthe wildtype.

It will be apparent to one of ordinary skill in the art that any DNApoint mutation can be enriched using oligonucleotides specific for themutation and prepared to encompass the mutated site.

Additionally, enrichment oligonucleotides can be prepared to enrich fora particular gene such as an oncogene regardless of the presence of amutation by preparing on oligonucleotide specific for the gene that doesnot encompass a site known to be involved in mutation related to thedevelopment of malignancy. Using such enrichment strategies, mutatednucleic acid is subsequently identified in the detection step of themethods of the invention, using mutation-specific oligonucleotides inhybridization assays, for example, or during a selective amplificationstep. For K-ras, enrichment oligonucleotides for nucleic acids encodingregions other than those containing codons 12, 13 or 61 are used toenrich a plasma, serum or whole blood sample for K-ras encoding nucleicacid sequences, both mutant and wildtype. Differentiation between mutantand wildtype fragment is then accomplished using detection methods suchas allele-specific oligonucleotide hybridization assays.

Advantageously, more than one enrichment oligonucleotide species can beused simultaneously or sequentially. A panel of such enrichmentoligonucleotides could include oligonucleotides specific for differentmutated sites in a particular oncogene, or several mutated oncogenes ina multiplex enrichment assay. Differential detection methods, such ashybridization with distinguishable detectable labels, are then used todetect the different mutated nucleic acids resulting from the enrichmentstep of the methods of the invention.

Additional enrichment oligonucleotides include those oligonucleotidesdisclosed in co-owned and co-pending U.S. Ser. No. 08/818,058, thedisclosure of which is explicitly incorporated herein.

Additional Nucleic Acid Extraction Methods

Alternate means of purification which may be used to obtain DNA fromserum or plasma, including selective retention on a size exclusioncolumn or similar matrix, salting-out method, and other guanidiniumthiocyanate extraction methods known in the art, including but notlimited to all methods described in co-owned and co-pending U.S. Ser.No. 08/818,058.

Alternatively, the plasma or serum may be prepared as described above,and one or several regions of DNA of interest may be enriched. This maybe performed by hybridization of nucleic acid in the heated plasma orserum solution or of extracted nucleic acid against a manufacturedstrand of nucleic acid that is complementary to the sequence of interest(an affinity capture approach; see Seeger et al., 1997, Biotechniques23: 512-7). Such “capturing” nucleic acids may consist ofoligonucleotides, DNA or RNA constructs, or peptide nucleic acids. Thecapture of the target DNA serves as a means of enriching the target atthe expense of other, non-hybridizing DNA that would otherwise competewith the target DNA during subsequent detection. The enrichmentprocedure occurs by hybridization at a low temperature undernonstringent conditions (see generally, Sambrook et al., 1989, MolecularCloning: A Laboratory Manual. (2nd ed.) Cold Spring Harbor LaboratoryPress, New York), followed by washing of the target-capture complex toremove unbound, competing DNA. The capture nucleic acid may be bound toa surface, such as the wall of a tube or a nylon or other membrane, orit may be bound to glass, magnetic or other beads that are incubatedwith the serum or plasma (Gelsthorpe et al., 1997, Biotechniques 22:1080-2; Rudi et al., 1997, Biotechiques 22: 506-11), thereby enablingseparation of the mutated nucleic acid alleles from wild-type nucleicacid alleles, for example but not limitation, by using a magnetic orelectric field, or by other methods well known to the art. A singleregion of interest may be examined in each sample, or several regionsmay be enriched for. By judicious choice of capturing nucleic acids,more than one target DNA may be captured simultaneously. It will beunderstood by one skilled in the art that this multiplexing approachwill speed analysis of samples, and may be performed in a relativelytumor-specific fashion, i.e., with a set of capture probes representinggenes that are mutated frequently in one type of tumor and infrequentlyin others. As an example, the genes TP53, KRAS, DCC and APC are oftenmutated in colorectal cancers and colorectal adenomas (precanceroustumors), while VHL and WT1 are more often mutated in renal cellcarcinomas. Thus, the identification of mutations in a series of genesor tumor-associated DNA may, beyond identifying the presence of amalignancy or premalignancy in a patient, serve to identify theparticular type of neoplasia by the particular set of genes ortumor-associated DNAs mutated or predict for the location of theneoplasia. Additionally, methods involving enrichment of the targetnucleic acid may be performed on unprocessed plasma, serum or wholeblood.

Alternatively, other means of enriching for the nucleic acid of interestin a plasma or serum sample may be used. For example, antibodiesdirected against any portion of the target DNA may serve to capture it,with subsequent identification of the presence or absence of mutations.However, several antibodies may be needed to capture the entire gene orDNA of interest if this is desired, since we and others have found thatDNA in plasma or serum often circulates in relatively small pieces, onthe order of several hundred base pairs in length. Such capturingantibodies may be raised in animals by immunization with the target DNAor fragments thereof, or may be purified from naturally occurringanti-DNA antibodies found in humans with rheumatologic conditions. Asexplained above, the capturing antibodies may be bound to tubes or wellsas in an ELISA, or may be bound to tubing through which a sample travelsprior to further analysis, as by gas chromatography/mass spectroscopy orhigh performance liquid chromatography or bound to beads. These meansand methods are provided by way of example and are not intended to belimiting.

Following the preparation of plasma or serum as described above, andpreferably following an amplification step, a detection step formutations in oncogenes or tumor-associated DNA is performed. It is thedetection of mutants that indicates the presence of one or moremutant-bearing cells within the patient. Moreover, as explained above,the pattern of mutants may be relatively specific for different types ofproliferative diseases. It bears stressing that this invention,comprising either precipitation of extracellular DNA from serum orplasma or preparation of serum, plasma or whole blood for analysiswithout precipitation of DNA followed by detection of mutations(including, but not limited to, point mutation, insertion, deletion andtranslocation) may be applied to detection of malignant neoplasms anddetection of precursor, premalignant conditions.

A mutated DNA detected in blood plasma or serum of a human may befurther characterized as being an acquired (somatic) mutation bydemonstrating the absence of the mutation in normal cells of the humanusing methods known in the art consisting of extraction of the DNA fromnormal cells, and amplifying for the mutated DNA of interest in a manneras to enable detection, wherein an absence of the mutated DNA in normalcells from the human indicates the mutation detected within blood plasmaor serum DNA to be an acquired mutation, and not an inherited or inbornmutation.

Detection of DNA sequence mutants may proceed by any of a number ofmethods known to those skilled in the art (Kilger et al., 1997, NucleicAcids Res. 25: 2032-4). The sequence may be detected directly by nucleicacid sequencing methods such as cycle sequencing (Sarkar et al., 1995,Nucleic Acids Res. 23: 1269-70) or direct dideoxynucleotide sequencing,in which some or all of the enriched DNA of interest that has beenharvested from plasma or serum is used as a template for sequencingreactions. An oligonucleotide primer or set of primers specific to thegene or DNA of interest is used in standard sequencing reactions.

Other methods of DNA sequencing, such as sequencing by hybridization,sequencing using a “chip” containing many oligonucleotides forhybridization (as, for example, those produced by Affymetrix Corp.,Ramsay et al., 1998, Nature Biotechnology 16: 40-44; Marshall et al.,1998, Nature Biotechnology 16: 27-31), sequencing by HPLC (DeDionisio etal., 1996, J Chromatogr A 735: 191-208), and modifications of DNAsequencing strategies such as multiplex allele-specific diagnostic assay(MASDA: Shuber et al., 1997, Hum. Molec. Genet. 6: 337-47), dideoxyfingerprinting (Sarkar et al., 1992, Genomics 13: 441-3; Blaszyk et al.,1995, Biotechniques 18: 256-60; Martincic et al., 1996, Oncogene 13:2039-44), and fluorogenic probe-based PCR methods (such as Taqman;Perkin-Elmer Corp.; Heid et al., 1996, Genome Res. 6: 986-94) andcleavase-based methods may be used. Alternatively, approaches thatdetect specific mutations of interest such as allele-specificamplification or restriction digest methods such as CARD (as disclosedin co-owned and co-pending U.S. Ser. No. 08/818,058, incorporated byreference) may be used singly or in combination to identifyextracellular mutant DNA (Zafiropoulos et al., 1997, Biotechniques 23:1104-1109). It will be clear to one skilled in the art that a variety ofsuitable methods for determining mutations at the DNA sequence levelwould suffice for the practice of this step of the inventive methods,and the methods mentioned herein are not intended to be comprehensive orlimiting, but merely to serve as examples.

Methods that detect mutations in DNA without precisely identifying themutated base or bases are also able to be used in this invention,inclusive of those methods of detection previously described in theapplication. Single strand conformation polymorphism (SSCP) analysis,for example, can identify variations from wildtype in a region of DNAwithout precisely defining the mutated base(s)(Orita et al., 1989, Proc.Natl. Acad. Sci. USA 86: 2766-70; Suzuki et al., 1990, Oncogene5:1037-43). Similarly, heteroduplex analysis (Glavac et al., 1995, Hum.Mutat. 6(4):281-7), denaturing gradient gel electrophoresis (Pellegataet al., 1992, Anticancer Res. 12: 1731-1736) and mismatch cleavageassays can identify a patient carrying a DNA mutation by analysis ofextracted or unextracted plasma or serum DNA. All of these proceduresrequire cleavage of the sample DNA with restriction endonucleases, oralternatively amplification by PCR or a similar amplification technique,to obtain uniform sized DNA fragments for gel electrophoresis analysis.

Detection of the target mutations can also be accomplished by means ofsignal amplification techniques. For example, the branched DNA assay(Chiron) uses a specific DNA probe to a target DNA (in this case, any ofthe oncogenes or tumor-associated DNAs of interest) to identify thepresence of the target (Urdea et al., 1993, AIDS 7: S11-4). The signalis amplified by means of modifications made to the probe which allowmany fluorescent detector DNA molecules to hybridize to a target nucleicacid. Similarly, oligonucleotide ligation assays may be used to amplifyand detect the mutation of interest from amplified or directly processedserum or plasma DNA (Benson et al., 1996, Thromb. Res. 83: 87-96). Anysignal amplification method may be used in the invention.

For the purposes of this invention, tumor-derived or associated DNAincludes but is not limited to DNA related to mutated oncogenes or othermutated DNA, a partial list of which includes H-ras, K-ras, N-ras,c-myc, her-2/neu, bcr-abl, fms, src, fos, sis, jun, bcl-2, bcl-2/IgH, orVHL (Von Hippel-Lindau gene), and DNA microsatellite alterations; DNArelated to tumor suppressor genes, a partial list of which includes p53,RB, MCC, APC, DCC, NF1, WT1; and hypermethylated DNA.

It will be understood that methods of detection and/or enrichment forextracellular mutated nucleic acid from blood or blood fractions asdescribed herein may similarly be applied as methods for detectionand/or enrichment of extracellular mutated nucleic acid present in otherbodily fluids, including particularly ascitic fluid, pleural effusions,pericardial effusions, sputum and bronchial secretions, breast fluidincluding secretion from the ducts and nipple of the breast, gastricsecretions, and fluid aspirated or drained from cystic or semi-cystictissues, wherein evaluation of the bodily fluid offers distinctdiagnostic advantage in humans in whom the presence of a disease isunknown.

Methods of Enrichment using an Endonuclease

Enrichment of the target or mutated nucleic acid, whereby theconcentration of the target nucleic acid is increased with respect towild-type or non-mutated nucleic acid, may be accomplished by using anendonuclease either before or during an amplification step, as disclosedin co-owned and co-pending U.S. Ser. No. 08/818,058, the disclosure ofwhich is explicitly incorporated herein. In preferred embodiments, theendonuclease may be BstNI, HinP I, or Msp I.

Therapeutic Applications

The extraction of extracellular DNA from plasma or serum permits furtheranalysis or other manipulation of that DNA, from which further clinicalutility is realized. In this optional step of the invention,extracellular nucleic acid is analyzed to define the characteristics orcomposition of the tumor or pre-neoplastic lesion from which the nucleicacid originates. Any of several methods may be used, dependent upon thedesired information, including nucleic acid sequencing, spectroscopyincluding proton NMR spectroscopy, biochemical analysis, and immunologicanalysis. In the preferred embodiment, such nucleic acid is cloned intoa plasmid vector, for example the pGEM-T vector plasmid (Promega,Madison, Wis.) and sequenced using a commercial kit such as Sequenase2.0 (USB, Cleveland, Ohio). Analysis to define the characteristics orcomposition of the extracellular nucleic acid, and thus thecharacteristics of the originating tissue, affords a wide array ofclinical utility, including the description, characterization, orclassification of the tumor, whether known or occult, by tissue oforigin, by type (such as premalignant or malignant), phenotype, orgenotype, or by description or characterization of tumor behavior,physiology and biochemistry. This information is useful to characterizethe lesion for tumor invasiveness, propensity to metastasize, andsensitivity or resistance to various therapies, thereby allowing theprediction of response to either ongoing or planned therapy and,further, allowing evaluation of prognosis. Comparison of thecharacteristics of extracellular nucleic acid to previous biopsy orsurgical specimens permits further evaluation of tumor heterogeneity orsimilarity in comparison to that specimen, and thus evaluation of tumorrecurrence or progression.

Following extraction of extracellular DNA from plasma or serum,complimentary ribonucleic acid (RNA) may be transcribed or manufacturedfrom the DNA. In a preferred embodiment, transcription of RNA isperformed by employing a primer with an RNA polymerase promoter regionjoined to the standard primer sequence for the DNA of interest. RNAcomplimentary to the DNA is then transcribed from the attached promoterregion. In an alternative method, extracellular nucleic acid is clonedinto an expression vector, and RNA complimentary to the DNA istranscribed. Furthermore, as an optional preferred embodiment, thecomplimentary RNA is used in an in vitro translation reaction tomanufacture tumor-associated or tumor-specific protein.

Extraction of extracellular nucleic acid from blood serum or plasma, andcharacterization, transcription of complimentary RNA, and translation totumor-associated or tumor-specific protein, provides significantutility, both in the delineation of those who might benefit fromtherapy, including chemoprevention or assignment of therapy, and in thedevelopment of tumor-specific therapies. Sequencing of extracellularnucleic acid or transcription of complementary RNA allows identificationor development of antisense compounds, including syntheticoligonucleotides and other antisense constructs appropriately specificto the extracellular DNA, such as by construction of an expressionplasmid such as by adapting the method of Aoki et al. (1995, Cancer Res.55: 3810-3816). Similarly, defining tumor characteristics allowsidentification of specific monoclonal antibody or vaccine therapiesappropriately specific to the extracellular DNA. Production ofcorresponding immunologic protein can be used in the development oftumor-specific monoclonal antibodies. Similarly, translated protein canbe used in tumor-specific vaccine development. Furthermore, theextracellular DNA permits a means of defining or allowing theconstruction of a DNA construct which may be used in vaccine therapy.

Of particular value, the invention allows the development andapplication of tumor-specific therapies even when only premalignanttumors, early cancers, or occult cancers are present. Thus, theinvention allows therapeutic intervention when tumor burden is low,immunologic function is relatively intact, and the patient is notcompromised, all increasing the potential for cure.

The invention also provides methods for transcribing RNA complementaryto the isolated extracellular nucleic acid from plasma or serum, as wellas methods for producing peptides and proteins (or fragments thereof)encoded thereby. Additional methods for using the peptide and proteinsas antigens for producing antibodies specific for the peptides andproteins encoded by the extracellular nucleic acids of the invention arealso provided. The isolated extracellular nucleic acids of the inventionare also used in methods for producing antisense oligonucleotides,either synthetically or using recombinant genetic methods, and the usethereof for affecting gene expression in a cell will be appreciated byone having ordinary skill in the art in view of the methods forisolating and identifying said extracellular nucleic acid providedherein. Vaccine production, as is understood by one with skill in theart, is also enabled using the methods of the invention.

The methods of the invention and preferred uses for the methods of theinvention are more fully illustrated in the following Examples. TheseExamples illustrate certain aspects of the above-described method andadvantageous results. These Examples are shown by way of illustrationand not by way of limitation.

EXAMPLE 1

Detection of Mutated K-ras DNA in the Plasma of Patients withoutClinically-Diagnosed Cancer using an Endonuclease-Based EnrichmentMethod

Colorectal adenomas are common premalignant neoplasms associated with arisk for development of colorectal cancer. Blood plasma wasprospectively obtained from 25 individuals who had K-ras mutatedcolorectal adenoma. None of the individuals were known to have cancer,nor did colonoscopy or other testing demonstrate cancer in anyindividual. Five to ten milliliters of blood was collected from eachindividual, and plasma was fractionated from the whole blood bycentrifugation at 400×g at room temperature for ten minutes.Extracellular DNA was then extracted from the plasma using a gelatinprecipitation method as previously described. The extracted plasma DNAwas amplified with a polymerase chain reaction assay which enriched formutated K-ras by employing simultaneous restriction digestion and PCRamplification utilizing a BstNI restriction endonuclease, the method ofwhich (CARD) is described in full in co-owned and co-pending U.S. Ser.No. 08/818,058, the disclosure of which is explicitly incorporatedherein. Mutations in the final digested amplified product wereidentified by agarose gel electrophoresis, and further prior to finalenzyme digestion by identification of altered bases using dot blothybridization. Following amplification, 5 μL of product were applied toa nylon membrane (MSI, Westboro, Mass.). Replicate blots were hybridizedto ³²P-radiolabelled oligonucleotides designed to identify pointmutations in positions 1 and 2 of codons 12 of the K-ras gene (MutaprobeHuman K-ras 12 set, Oncogene Science, Uniondale, N.Y.). Hybridizationand wash conditions were as specified by the membrane's manufacturer.Blots were exposed to x-ray film for 10 hours at −80° C. Mutated K-rasDNA was detected in the plasma of 20 individuals. Sequence analysisdemonstrated that the altered base mutation found in the adenoma wassimilarly demonstrable in the plasma of the individuals.

This example demonstrates that an enrichment-based assay enablesdetection of mutated nucleic acid in individuals withoutclinically-diagnosed cancer, further enables detection of the presenceof premalignant tissue including colorectal adenoma; further enablesidentification of a predictive risk factor; further enables sequenceidentification of the mutated DNA in blood and further predicts for thesequence of the mutated DNA in tissue.

EXAMPLE 2

Detection of Mutated P53 DNA in the Plasma of Individuals withoutClinically-Diagnosed Cancer using an Enrichment Assay

P53 mutations are common oncogene mutations in both malignant andpremalignant tumors. Plasma was obtained prospectively from individualsundergoing colonoscopy and assayed for plasma DNA containing DNA speciesof P53 mutations at codons 175 and 248. Five to ten milliliters of bloodwere collected in EDTA-coated siliconized glass vacutainer tubes fromeach individual assayed. Plasma was fractionated from whole blood bycentrifugation at 400×g at room temperature for ten minutes.Extracellular DNA was then extracted from plasma using a gelatinprecipitation extraction as previously described. 7 μL of thegelatin-precipitated DNA were then used in an amplification reaction inwhich P53 DNA was enriched using a restriction endonuclease.Oligonucleotide primers were designed to perform a hemi-nestedamplification of a portion of exon 5 for codon 175, wherein the firstprimer (P53-1) is 5′-GCAGTCACAGCACATGACG-3′ (SEQ ID No. 7); the secondprimer (P53-2) is 5′-AATCAGAGGCCTGGGGAC-3′ (SEQ ID No. 8); and the thirdprimer (P53-3) is 5′-GGGCCAGACCTAAGAGCAAT-3′ (SEQ ID No. 9). A reactionmixture consisting of 1×Taq polymerase (Fisher, Pittsburgh, Pa.) in avolume of 50 μL was prepared. This mixture was amplified by polymerasechain reaction under mineral oil for 20 cycles with denaturation at 94°C. for 1 minute, annealing at 58° C. for 90 seconds, and extension at72° C. for 90 seconds. This was followed by a CARD step of combinedamplification and restriction digestion, in which 5 μL of the initialamplification product was transferred to a new reaction mix identical tothe K-ras mixture previously described for CARD in co-pending andco-owned U.S. Ser. No. 08/818,058, the disclosure of which is explicitlyincorporated herein, except for the use of 10 picomoles each of primersP53-1 and P53-2 in place of K-ras primers, and the substitution of 8units of restriction enzyme HinP1 I (New England BioLabs, Beverly,Mass.) for BstNI. Cycling parameters were as for the CARD K-rasamplification. A final digestion with HinP1 I was performed prior toidentification of mutants by agarose gel electrophoresis. To detectcodon 248 P53 mutations a similar CARD assay was performed using primers(P53-4): 5′-TTGGGCCGGTGTTATCTC-3′ (SEQ ID No. 10); and (P53-5):5′-ATGGTGCGGATGGGCCTC-3′ (SEQ ID No. 11), designed to amplify a portionof exon 7. Mutations at codon 248 were enriched and amplified in thesame manner as described above for codon 175, except substituting HinP1I with Msp I (New England BioLabs, Beverly, Mass.). Mutations in thefinal digested amplified product were identified by agarose gelelectrophoresis.

Extracellular mutated P53 DNA was demonstrated in the plasma of 3individuals, none of whom had clinically-diagnosed cancer, including oneindividual having a codon 175 P53 mutation, and 2 individuals having acodon 248 P53 mutation. Colonoscopy demonstrated a premalignant lesion(an adenoma) in each of two of these individuals, and a proliferativedisease (a hyperplastic polyp) in the third individual.

This example further demonstrates that extracellular mutated nucleicacid can be detected in the blood of humans without clinically-diagnosedcancer; further that enrichment of the target nucleic acid may beaccomplished using a restriction endonuclease; further that mutated P53DNA may be detected in the blood of humans without clinically-diagnosedcancer; further that mutated P53 DNA may be enriched from the blood ofhumans without clinically-diagnosed cancer; further that mutated P53 DNAmay be detected in the blood of individuals with premalignant neoplasmsor proliferative disease; further that P53 DNA provides a predictiverisk factor for neoplastic disease. While the example demonstratesdetection of P53 mutations at codon 175 and 248, P53 mutations at othercodons may similarly be detected in blood by substituting theappropriate primers and restriction enzyme.

EXAMPLE 3

Enhanced Identification of Humans with Premalignant Neoplasms using aMultiplex Assay or Assay Panel Targeting Differing Mutated Oncogenes

Since individuals with premalignant neoplasms and conditions may harbordiffering mutated oncogenes or differing DNA microsatellite alterations,evaluating blood for a number of differing mutated oncogenes ortumor-associated nucleic acids are thereby more likely to enableidentification of those individuals harboring neoplasms, therebyincreasing overall sensitivity in identification of said individuals.

Plasma was evaluated by a panel of assays which detect mutated K-ras DNA(by the method provided in example 1), and mutated P53 DNA (by themethod provided in example 2). Plasma from individuals withoutclinically-diagnosed cancer shown to harbor colorectal adenoma wereevaluated for both extracellular mutated K-ras and mutated P53 DNA.Mutated K-ras but not P53 DNA was demonstrated in the plasma from 20individuals having K-ras mutated adenoma. However, mutated P53 but notK-ras DNA was additionally demonstrated in the plasma from 2 individualshaving wild-type ras adenoma. Together, the panel of both K-ras and P53assays thereby enabled increased identification of individuals withcolorectal adenoma. Increasing the number of mutated oncogenes andtumor-associated nucleic acids detected by the assay array or panel (forexample but not limitation by including an APC assay as provided inexample 4) thereby increases the ability to identify individualsharboring premalignant neoplasms, thereby increasing the utility of theassay.

EXAMPLE 4

Detection of an APC Gene Mutation in Plasma from an Individual with aColorectal Neoplasm

APC gene mutations are common mutations in colorectal premalignant andmalignant neoplasms. APC mutations may be demonstrated in blood ortissue by the method as follows: Three overlapping sets ofoligonucleotide primers are prepared to the mutation cluster region inexon 15 of the APC gene, which are estimated to account for over 60% ofall APC mutations in colorectal neoplasms. The primer sequences are:APC-1, 5′-TCCACACCTTCATCTAATGCC-3′;  (SEQ ID No. 12)APC-2, 5′-CATTCCACTGCATGGTTCAC-3′;  (SEQ ID No. 13)APC-3, 5′-CTGAAGATCCTGTGAGCGAA-3′;  (SEQ ID No. 14)APC-4, 5′-TCAGGCTGGATGAACAAGAA-3′;  (SEQ ID No. 15)APC-5, 5′-CTTCGCTCACAGGATCTTCA-3′;  (SEQ ID No. 16)APC-6, 5′-TTTGAGAGTCGTTCGATTGC-3′.  (SEQ ID No. 17)

DNA extracted from plasma, serum, whole blood, or tissue is subjected topolymerase chain reaction amplification with 1 picomole each of eitherprimers APC-1 and APC-2, or primers APC-3 and APC-4 for 25 cycles at adenaturing temperature of 94° C. for 60 seconds, annealing at 51° C. for90 seconds, and extension at 72° C. for 90 seconds each cycle. Thereaction mixture consists of 1×Taq buffer, 1.5 mM MgCl₂, 200 micromolardNTPs, and 1 unit Taq polymerase (Fisher, Pittsburgh, Pa.) in a volumeof 50 μL. Hemi-nested PCR amplification is then performed on 5 μL of thefirst amplification product, wherein primers APC-1 and APC-2 arereplaced with primers APC-1 and APC-5; and in a separate reactionprimers APC-1 and APC-2 replaced with primers APC-2 and APC-3; and in aseparate reaction primers APC-3 and APC-4 replaced with primers APC-6and APC-4. Reaction conditions for the second round of amplification arethe same as for the first, except that 10 picomoles of each primer areused, and the cycle number is 35. The PCR products are detected byagarose gel electrophoresis, bands are excised from the gel and DNAisolated using the GeneClean kit (Bio101) according to themanufacturer's instructions. DNA is then asymmetrically reamplified withone primer at 100 picomoles and the other at 2 picomoles for eventualcycle sequencing, wherein primer pairs are: APC-1 and APC-5; APC-2 andAPC-3 APC-4 and APC-6. The asymmetric amplification is performed asdescribed in McCabe (1990, PCR Protocols. A guide to methods andapplications (Innis, Gelfand, Sninsky, & White, eds.) Academic Press,pp. 76-83) using approximately 1.4 ng of each PCR product and annealingat 51° C. Cycle sequencing is then performed as described in Brow (1990,PCR Protocols. A guide to methods and applications (Innis, Gelfand,Sninsky, & White, eds.) Academic Press, pp. 189-196) using the limitingprimer labeled with ³²P.

5-10 mL of blood was collected in an EDTA-coated siliconized glassvacutainer tube from an individual with colorectal clinically-diagnosedcancer whose tumor was known to harbor an APC mutation consisting of afive base deletion at 3961-3965. Plasma was fractionated from wholeblood by centrifugation at 400×g at room temperature for ten minutes.DNA was then extracted from 200 μL of plasma using the High-Pure ViralNucleic Acid kit (Boehringer Mannheim). The extracted DNA was initiallyamplified as described above using primers APC-1 and APC-7(5′-TGCTGGATTTGGTTCTAGGG-3′; SEQ ID No. 18), then reamplified withprimers APC-7 and APC-8 (5′-TCAGACGACACAGGAAGCAG-3′; SEQ ID No. 19) asin the hemi-nested reactions described above. The PCR products wereelectrophoresed through a 5% agarose gel and examined by ethidiumbromide staining. The appropriate region of the gel was excised andprepped by the GeneClean, and this DNA then cycle sequenced as describedabove, thereby confirming an APC mutation in the plasma DNA.

Plasma from this individual having APC mutated plasma DNA was furtherevaluated for the presence of mutated K-ras DNA (by the method describedin example 1), and mutated P53 DNA (by the method described in example2). Plasma was negative for both mutated K-ras and mutated P53 DNA inthis individual.

This example demonstrates that mutated APC DNA may be detected in blood.Further, detection of mutated APC in blood is indicative of the presenceof mutated tissue, and provides a predictive risk factor for neoplasticdisease. The example demonstrates that detection of mutated APC in bloodis particularly advantageous in predicting for neoplastic disease of agastrointestinal origin, and particularly a colorectal neoplasticdisease.

It will be understood that enriching the extracted DNA for APC bymethods as described in the invention will further increase thesensitivity of the assay for detection of mutated APC DNA in blood. Itis further shown by this example that it is particularly advantageous toinclude methods detecting mutated APC within a multiplexed assay or anoncogene or tumor-associated nucleic acid assay panel or array, whereinit is particularly advantageous to assay blood plasma or serum formutated APC in addition to mutated K-ras and/or mutated P53. It is wellrecognized that APC mutations occur early and commonly in premalignantneoplasms such as colorectal adenoma, thus it is particularly evidentthat it is advantageous to detect mutated APC in the blood of humanswithout clinically-diagnosed cancer, as to predict for premalignantdisease or risk for neoplastic disease, and to further indicate the needfor additional diagnostic testing including but not limited toendoscopy, colonoscopy, sigmoidoscopy, radiologic imaging,radionucleotide scanning, ultrasonography, PET scanning, or furtherevaluation of organ or site specific bodily fluids or stool, whereinbodily fluid includes but is not limited to that collected by drainage,aspiration, direct sampling, and lavage.

EXAMPLE 5

Detection of mutated K-ras DNA in the Plasma of a Human withoutClinically-Diagnosed Cancer having a Hyperplastic ProliferativeCondition

Hyperplastic colorectal polyps are non-neoplastic proliferativeconditions. Blood was prospectively examined from a human found uponcolonoscopy and polypectomy to have a K-ras mutated hyperplastic polypbut not to have cancer. The method of examination was identical to themethod provided in example 1. Mutated K-ras DNA was similarlydemonstrated in the plasma from the human. This example demonstratesthat the methods of the invention enable detection of mutated DNA in theblood of individuals having hyperplastic tissue harboring mutated DNA.

EXAMPLE 6

Detection of Mutated K-ras DNA in the Plasma of a Human with an In-Situ(Non-Invasive) Carcinoma

In-situ carcinomas are early cancers which by definition are localizedand non-invasive. A human undergoing a colonoscopy was found to have aK-ras mutated in-situ colorectal carcinoma. Plasma was prospectivelyobtained from the human and examined using the methods identical tothose provided in example 1. Mutated K-ras DNA was similarly detected inthe plasma from the human. This example demonstrates that the methods ofthe invention enable detection of mutated DNA in blood from humanshaving localized, non-invasive or pre-invasive, in-situ malignancy whenthe malignancy harbors mutated DNA.

EXAMPLE 7

Prophetic Examples of the Use of the Assays of the Invention

The following example is illustrative of potential clinical uses for theassays of the invention.

Case 1

A 70 year old woman with a long history of cigarette smoking visits herdoctor because she is concerned about her risk of developing lungcancer. A sample of blood is drawn and plasma is prepared. An aliquot ofthe plasma is incubated with metal beads containing oligonucleotidescomplementary to exons of the TP53 tumor suppressor gene bound to theirsurface. The beads are drawn to the side of the tube with a magnet, andthe tube is washed several times with water. The tube is then heated torelease the affinity-captured TP53 DNA fragments, and the solutioncontaining these fragments is applied to a sequencing chip useful fordetecting mutations in the TP53 gene (commercially available fromAffymetrix). The machine designed to read the sequencing chip detects adeletion of codon 158 of the TP53 gene. Confirmation of the presence ofthe TP53 mutation is obtained from a sputum sample, and it is concludedthat the patient is at high risk for development of lung cancer. She isclosely followed by CXR, spiral CT scan, and bronchoscopy, and thepatient has blood plasma regularly monitored by a version of theinvention adapted specifically for her TP53 mutation. After enrichmentof the plasma for the exon carrying the codon 158 mutation using metalbeads followed by amplification, a quantitative assay for the presenceof the mutation is used to determine the amount of mutant present in theplasma. For one year, the patient has no detectable mutant DNA,following which a steady rise in the amount of mutant DNA is noted oversix months. No clinical or radiographic evidence of disease is presentat this time. The patient begins a P53-directed chemopreventive therapyand after a successful course remains free of disease while beingmonitored by the codon 158 assay.

This example further illustrates the use of the invention in aquantitative fashion, in this case using the affinity-capture version ofthe invention. A tumor suppressor gene frequently altered in lung cancer(TP53) is enriched from the plasma and used as the substrate for aquantitative portion of the invention. This mutant can then be followedand intervention begun early, when chemopreventive therapy is mosteffective.

This clinical vignette is meant as an example of the uses to which theinvention may be put, and is not meant in any way to be limitations uponthe range or type of assays or extracellular mutant DNAs detectable inthe plasma or serum by the invention.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

1. A method for determining an acquired a predictive risk factor for anon-hematologic disease in a human without cancer having a , whereinsaid non-hematologic disease is a premalignancy that is an adenoma, ornon-hematopoietic dysplastic or hyperplastic cells or tissue, the methodcomprising the steps of: a. purifying extracellular nucleic acid fromblood from a the human without cancer to prepare extracted extracellularnucleic acid containing DNA encoding a mutated gene or fragment thereof;and concurrently or sequentially, b. enriching the extractedextracellular nucleic acid for the mutated gene DNA or a fragmentthereof, wherein the mutated gene DNA or a fragment thereof isconcentrated or isolated from the remaining extracted extracellularnucleic acid; c. amplifying a mutated gene DNA or a fragment thereof, oramplifying a signal from the enriched mutated DNA or a fragment thereof;and d. detecting the product of the amplified mutated gene DNA or theproduct of its amplified fragment, or the amplified signal of themutated DNA or the amplified signal of a fragment thereof, whereby saiddetection determines a predictive risk factor for a non-hematologicdisease in the human without cancer, wherein said non-hematologicdisease is a premalignancy that is an adenoma, or non-hematopoieticdysplastic or hyperplastic cells or tissue.
 2. A method according toclaim 1, wherein the product of the amplified mutated gene DNA isdetected using a detection method that is gel electrophoresis, singlestrand conformation polymorphism, heteroduplex analysis, denaturinggradient gel electrophoresis, mismatch cleavage assay, immunologicaldetection methods, nucleic acid hybridization, Southern blot analysis,electrochemiluminescence, reverse dot blot detection, orhigh-performance liquid chromatography.
 3. The method of claim 1 whereinextracellular nucleic acid is purified from the plasma or serum fractionof blood.
 4. The method of claim 1 wherein the enriched mutated gene DNAor a fragment thereof of subpart (b) is amplified in subpart (c) usingan amplification method that is polymerase chain reaction, ligase chainreaction, boomerang DNA amplification, Q-beta replication,transcription-based amplification, isothermal nucleic acid sequencebased amplification, self-sustained sequence replication assay, stranddisplacement activation, or cycling probe technology.
 5. A method ofclaim 1 wherein mutated gene DNA or a fragment thereof is enriched insubpart (b) through an endonuclease-mediated restriction digestion.
 6. Amethod of claim 1 wherein mutated gene DNA or a fragment thereof isenriched in subpart (b) by hybridization of the mutated gene DNA or afragment thereof to an oligonucleotide to form a hybridized complex. 7.A method according to claim 6 wherein the hybridized complex is furthersubjected to a magnetic field.
 8. A method according to claim 6 whereinthe hybridized complex is further subjected to an electric field.
 9. Amethod according to claim 6 wherein the endonuclease is BstNI, HinP1 I,or Msp I.
 10. A method according to claim 1, wherein the mutated DNAencodes a mutated oncogene or fragment thereof.
 11. A method accordingto claim 10, wherein the mutated oncogene is mutated K-ras.
 12. A methodaccording to claim 10, wherein the mutated oncogene is mutated APC. 13.A method according to claim 1, wherein the mutated DNA is DNA having amicrosatellite alteration.
 14. A method according to claim 1, whereinthe non-hematologic disease is colorectal adenoma, cervical dysplasia,atypical squamous metaplasia of the lung, bronchial dysplasia, atypicalhyperplasia of the breast, prostatic intraepithelial neoplasia, atypicalendometrial hyperplasia, dysplastic nevi of the skin, or Barrett'sesophagus.
 15. The method of claim 1, further comprising the steps of:e) demonstrating that the mutated gene DNA is absent in normal cellsfrom the human, thereby indicating that the predictive risk factor is anacquired predictive risk factor.
 16. A method for enriching purifiedextracellular DNA for extracellular mutated DNA or a fragment thereof inblood of a human without cancer having a premalignancy that is anadenoma, or non-hematopoietic dysplastic or hyperplastic cells ortissue, the method comprising the steps of: a. purifying extracellularnucleic acid from blood of a human without cancer to prepareextracellular nucleic acid containing a mutated DNA species or fragmentthereof, and concurrently or sequentially b. hybridizing anoligonucleotide that is complementary to the mutated DNA or a mutatedDNA fragment to produce a hybridized product of mutated DNA or afragment thereof; and c. separating the hybridized product of mutatedDNA or a fragment thereof from the remaining non-hybridizedextracellular nucleic acid, thereby enriching the purified extracellularDNA for the mutated DNA or a fragment thereof.
 17. The method of claim16 wherein the extracellular nucleic acid is purified from the plasma orserum fraction of blood.
 18. A method according to claim 16 wherein theoligonucleotide is further bound to a non-biologic surface.
 19. A methodaccording to claim 16 wherein in subpart (c) the hybridized product issubjected to a magnetic field.
 20. A method according to claim 16wherein in subpart (c) the hybridized product is subjected to anelectric field.
 21. A method according to claim 16, wherein the mutatedDNA is mutated oncogene DNA.
 22. A method according to claim 21, whereinthe mutated oncogene is mutated K-ras.
 23. A method according to claim21, wherein the mutated oncogene is mutated APC.
 24. A method accordingto claim 21, wherein the mutated oncogene is mutated P53.
 25. A methodaccording to claim 16, wherein the mutated DNA is DNA having amicrosatellite alteration.
 26. A method of identifying a human having adisease or premalignancy without premalignant condition in a humanwherein the human does not have clinical symptoms by detecting amutation in DNA from blood or a blood fraction from the human, whereinthe mutation is an acquired mutation that is associated with the diseaseor premalignancy premalignant condition, and wherein the disease orpremalignant condition is colorectal adenoma, cervical dysplasia,atypical squamous metaplasia of the lung, bronchial dysplasia, atypicalhyperplasia of the breast, prostatic intraepithelial neoplasia, atypicalendometrial hyperplasia, dysplastic nevi of the skin, or Barrett'sesophagus; the method comprising the steps of: a. purifyingextracellular nucleic acid from blood or a blood fraction from a thehuman without cancer to prepare extracted extracellular nucleic acidcontaining said mutated DNA; b. amplifying the mutated DNA, oralternatively amplifying a signal from the mutated DNA; and c. detectingthe product of the amplified mutated DNA or the amplified signal of themutated DNA, wherein the human is identified as having the disease orpremalignancy premalignant condition.
 27. A method according to claim26, wherein the evaluation of blood or a blood fraction from a human forsaid mutation assists in the identification of said disease orpremalignant condition.
 28. A method according to claim 26, whereinextracellular nucleic acid is purified from the plasma or serum fractionof blood.
 29. A method according to claim 26, further comprising thestep of performing a diagnostic test that is colonoscopy, sigmoidoscopy,endoscopy, bronchoscopy, radiologic imaging, ultrasonography,radionucleotide imaging, PET scanning, and evaluation of organ specificbodily fluid, stool, or lavage fluid.
 30. A method according to claim26, wherein the mutated DNA is a DNA microsatellite alteration.
 31. Themethod of claim 26, wherein the mutated DNA of subpart (a) is amplifiedin subpart (b) using an amplification method that is polymerase chainreaction, ligase chain reaction, boomerang DNA amplification, Q-betareplication, transcription-based amplification, isothermal nucleic acidsequence based amplification, self-sustained sequence replication assay,strand displacement activation, or cycling probe technology.
 32. Themethod of claim 26, wherein the product of the amplified mutated DNA isdetected using a method that is gel electrophoresis, single strandconformation polymorphism, heteroduplex analysis, denaturing gradientgel electrophoresis, mismatch cleavage assay, immunological detectionmethods, nucleic acid hybridization, Southern blot analysis,electrochemiluminescence, reverse dot blot detection, orhigh-performance liquid chromatography.
 33. A method according to claim26, wherein prior to amplification in subpart (b), the mutated DNA insubpart (a) is enriched, wherein the mutated DNA is concentrated orisolated from the remaining extracted nucleic acid.